Method for screening for possible presence of cancer

ABSTRACT

A new family of tumor rejection antigen precursors, and the nucleic acid molecules which code for them, are disclosed. These tumor rejection antigen precursors are referred to as DAGE tumor rejection antigen precursors, and the nucleic acid molecules which code for them are referred to as GAGE coding molecules. Various diagnostic and therapeutic uses of the coding sequences and the tumor rejection antigens, and their precursor molecules are described.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/809,999filed Apr. 9, 1997, which is the 371 national stage filing of PCTapplication US95/12116, filed Sep. 21, 1995, which is a continuation inpart of application Ser. No. 08/316,231, filed Sep. 30, 1994, thedisclosure of which is incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a nucleic acid molecule which code for a tumorrejection antigen precursor. More particularly, the invention concernsgenes, whose tumor rejection antigen precursor is processed, inter alia,into at least one tumor rejection antigen that is presented by HLA-A24molecules. The tumor rejection antigen precursor, or "TRAP" may beprocessed into additional peptides presented by other MHC molecules,such as HLA-A1 and its alleles, HLA-A2, HLA-Cw*1601, HLA-B44, and soforth. The genes in question do not appear to be related to other knowntumor rejection antigen precursor coding sequences, are expressed on avariety of tumors and, with the exception of testis, ovary andendometrial cells, are not expressed by normal cells.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T lymphocyte, or "T cell" response. This responserequires that T cells recognize and interact with complexes of cellsurface molecules, referred to as human leukocyte antigens ("HLA"), ormajor histocompatibility complexes ("MHCs"), and peptides. The peptidesare derived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See in this regard Male et al.,Advanced Immunology (J.P. Lipincott Company, 1987), especially chapters6-10. The interaction of T cells and HLA/peptide complexes isrestricted, requiring a T cell specific for a particular combination ofan HLA molecule and a peptide. If a specific T cell is not present,there is no T cell response even if its partner complex is present.Similarly, there is no response if the specific complex is absent, butthe T cell is present. This mechanism is involved in the immune system'sresponse to foreign materials, in autoimmune pathologies, and inresponses to cellular abnormalities. Much work has focused on themechanisms by which proteins are processed into the HLA bindingpeptides. See, in this regard, Barinaga, Science 257: 880 (1992);Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257:927 (1992); Latron et al., Science 257: 964 (1992).

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs cytolytic Tlymphocytes, or "CTLs" hereafter. The genes are said to code for "tumorrejection antigen precursors" or "TRAP" molecules, and the peptidesderived therefrom are referred to as "tumor rejection antigens" or"TRAs". See Traversari et al., Immunogenetics 35: 145 (1992); van derBruggen et al., Science 254: 1643 (1991), for further information onthis family of genes. Also, see U.S. patent application Ser. No.807,043, filed Dec. 12, 1991, now U.S. Pat. No. 5,342,774, incorporatedby reference in its entirety. The "MAGE" family of tumor rejectionantigen precursors is disclosed in this patent.

In U.S. patent application Ser. No. 938,334, now U.S. Pat. No. 5,405,940the disclosure of which is incorporated by reference, it is explainedthat the MAGE-1 gene codes for a tumor rejection antigen precursor whichis processed to nonapeptides which are presented by the HLA-A1 molecule.The nonapeptides which bind to HLA-A1 follow a "rule" for binding inthat a motif is satisfied. In this regard, see e.g. PCT/US93/07421; Falket al., Nature 351: 290-296 (1991); Engelhard, Ann Rev. Immunol. 12:181-207 (1994); Ruppert et al., Cell 74: 929-937 (1993); Rotzschke etal., Nature 348: 252-254 (1990); Bjorkman et al., Nature 329: 512-518(1987); Traversari et al., J. Exp. Med. 176: 1453-1457 (1992). Thereference teaches that given the known specificity of particularpeptides for particular HLA molecules, one should expect a particularpeptide to bind to one HLA molecule, but not to others. This isimportant, because different individuals possess different HLAphenotypes. As a result, while identification of a particular peptide asbeing a partner for a specific HLA molecule has diagnostic andtherapeutic ramifications, these are only relevant for individuals withthat particular HLA phenotype. There is a need for further work in thearea, because cellular abnormalities are not restricted to oneparticular HLA phenotype, and targeted therapy requires some knowledgeof the phenotype of the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 andincorporated by reference, the fact that the MAGE-1 expression productis processed to a second TRA is disclosed. This second TRA is presentedby HLA-Cw*1601 molecules. The disclosure shows that a given TRAP canyield a plurality of TRAs, each of which will satisfy a motif rule forbinding to an MHC molecule.

In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, andincorporated by reference herein teaches that tyrosinase, a moleculewhich is produced by some normal cells (e.g., melanocytes), is processedin tumor cells to yield peptides presented by HLA-A2 molecules.

In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993, andincorporated by reference in its entirety, a second TRA, not derivedfrom tyrosinase is taught to be presented by HLA-A2 molecules. The TRAis derived from a TRAP, but is coded for by a non-MAGE gene. Thisdisclosure shows that a particular HLA molecule may present TRAs derivedfrom different sources.

In U.S. patent application Ser. No. 08/079,110, filed Jun. 17, 1993 andincorporated by reference herein, an unrelated tumor rejection antigenprecursor, the so-called "BAGE" precursor is described. The BAGEprecursor is not related to the MAGE family.

In U.S. patent applications Ser. No. 08/096,039 and Ser. No. 08/250,162,both of which are incorporated by reference, non-related TRAP precursorGAGE is also disclosed.

The work which is presented by the papers, patent, and patentapplications cited supra deals, in large part, with the MAGE family ofgenes, and the unrelated BAGE and GAGE genes. It has now been found,however, that additional tumor rejection antigen precursors areexpressed by cells. These tumor rejection antigen precursors arereferred to as "DAGE" tumor rejection antigen precursors. They do notshow homology to the MAGE family of genes, the BAGE gene, or the GAGEgene. Thus the present invention relates to genes encoding such TRAPs,the tumor rejection antigen precursors themselves as well asapplications of both.

What further characterizes the DAGE tumor rejection antigen precursorsis that their expression by tumor cells is much more widespread than theother tumor rejection antigen precursors described previously. This isproven infra. Yet, the expression of the family by normal cells is againlimited to testis, ovary and endometrial cells. Thus, a much moregeneral means of assaying for the presence of transformed cells isavailable than previously. This will be seen by way of the examples.

The invention is elaborated upon further in the disclosure whichfollows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 describes, collectively, ⁵¹ Cr release, cell lysis studies. Inparticular:

FIG. 1A shows lysis of cell line LB33-MEL.B-1;

FIG. 1B shows lysis of LB33 B cells transformed by EBV. These areautologous cells.

FIG. 1C shows lysis studies on NK target K562. In each case, theeffector cells were CTL clone LB33-CTL-269/17.

FIGS. 2A and 2B present studies on the inhibition of lysis by cytolyticT cells in the presence of an anti-HLA-A24 monoclonal antibody. Thestudies were carried out in the presence or absence of 30 fold dilutionsof culture medium of a hybridoma producing the HLA-A24 specificmonoclonal antibody.

FIG. 3 shows the result of lysis experiments following transfection ofLB804-ALL cells with the sequence Hi2.

FIGS. 4A, 4B and 4C show the results obtained in a TNF release assayusing CTL 269/17. The stimulator cells were either LB33-MEL.B-1, COS-7cells, COS-7 cells tranfected with a cDNA sequence coding for HLA-A24,or COS-7 cells transfected with both cDNA sequence coding for HLA-A24,and cDNA coding for a tumor rejection antigen precursor in accordancewith this invention.

FIG. 5 compares induced lysis using various peptides derived from DAGE.

FIG. 6 shows the expression of DAGE in various tissue samples.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Melanoma cell line LB33-MEL.B was derived from a metastasis of patientLB33, using standard techniques. Tumor cells were then cloned bylimiting dilution, resulting in clone LB33-MEL.B-1, used hereafter.

Samples containing mononuclear blood cells (which include lymphocytes)were taken from patient LB33. Samples of clone LB33-MEL.B-1 werecontacted to the mononuclear blood cell samples. The mixtures wereobserved for lysis of the LB33-MEL.B-1 cells, this lysis indicating thatcytolytic T cells ("CTLs") specific for a complex of peptide and HLAmolecule presented by the cells were present in the sample.

The lysis assay employed was a chromium release assay following Herin etal., Int. J. Cancer 39:390-396 (1987), the disclosure of which isincorporated by reference. The assay, however, is described herein. Thetarget melanoma cells were grown in vitro. Prior to labelling, thesecells were incubated for 48 hours, in the presence of 50 U/ml of IFN-γto increase the expression of HLA Class I molecules. The cells were thenresuspended at 10⁷ cells/ml in DMEM, supplemented with 10 mM HEPES and30% FCS (i.e. fetal calf serum), and incubated for 45 minutes at 37° C.with 200 μCi/ml of Na(⁵¹ Cr)O₄. Labelled cells were washed three timeswith DMEM, supplemented with 10 mM Hepes. These were then resuspended inDMEM supplemented with 10 mM Hepes and 10% FCS, after which 100 ulaliquots containing 10³ cells, were distributed into 96 wellmicroplates. PBL containing samples were added in 100 ul of the samemedium, and assays were carried out in duplicate. Plates werecentrifuged for 4 minutes at 100 g, and incubated for four hours at 37°C. in a 5.5% CO₂ atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant werecollected and counted. Percentage of ⁵¹ Cr release was calculated asfollows: ##EQU1## where ER is observed, experimental ⁵¹ Cr release, SRis spontaneous release measured by incubating 10³ labeled cells in 200ul of medium alone, and MR is maximum release, obtained by adding 100 ul0.3% Triton X-100® to target cells.

Those mononuclear blood cell samples which showed high CTL activity wereexpanded and cloned via limiting dilution, and were screened again,using the same methodology.

The same method was used to test target K562 cells. When EBV-B cellswere used, the only change was the replacement of DMEM medium by Hank'smedium, supplemented with 5% FCS.

These experiments led to isolation of CTL clone LB33-CTL-269/17 frompatient LB33. As FIGS. 1A-1C indicate, this CTL clone lysed LB33-MEL.B-1tumor cells, but not EBV transformed B cells of patient LB33, nor K562cells. When the target cells were incubated with a monoclonal antibodyspecific to HLA-A24, lysis was inhibited, suggesting that any TRApeptide involved is presented by HLA-A24. FIG. 2 shows these results.

A second CTL clone, referred to as LB33-CTL-269/1, lysed LB33-MEL.B-1but not EBV-B transformed B cells nor K562, thus suggesting that thesame target antigen was recognized. Lysis by clone LB33-CTL-269/1 wasalso inhibited by the anti-HLA-A24 monoclonal antibody.

EXAMPLE 2

Having identified the presenting MHC molecule as HLA-A24, studies werecarried out to identify the coding sequence for the protein molecule,referred to hereafter as the "tumor rejection antigen precursor" or"TRAP" molecule which was the source of the presented peptide.

To do this, total RNA was isolated from cell line LB33-MEL.B-1. The mRNAwas isolated using an oligo-dT binding kit, following well recognizedtechniques. Once the mRNA was secured, it was transcribed into cDNA,again using standard methodologies. The cDNA was then ligated to EcoRIadaptors and cloned into the EcoRI site of plasmid pcDNA-I/Amp, inaccordance with manufacturer's instructions. The recombinant plasmidswere then electroporated into DH5α E. coli (electroporation conditions:1 pulse at 25 μfarads, 2500 V).

The transfected bacteria were selected with ampicillin (50 μg/ml), andthen divided into 400 pools of 100 clones each. Each pool representedabout 50 different cDNAs, as analysis showed that all plasmids containedan insert and cloning was not directional. Each pool was amplified tosaturation, and plasmid DNA was isolated via alkaline lysis, potassiumacetate precipitation and phenol extraction, following Maniatis et al.,in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.,1982). Cesium gradient centrifugation was not used.

EXAMPLE 3

The amplified plasmids were then transfected into eukaryotic cells.Samples of COS-7 cells were seeded, at 15,000 cells/well into tissueculture flat bottom microwells, in Dulbecco's modified Eagles Medium("DMEM") supplemented with 10% fetal calf serum. The cells wereincubated overnight at 37° C., medium was removed and then replaced by30 μl/well of DMEM medium containing 10% Nu serum, 400 μg/mlDEAE-dextran, 100 μM chloroquine, 100 ng of plasmid pcDNA-I/Amp-A2A and100 ng of DNA of a pool of the cDNA library described supra. PlasmidpcDNA-I/Amp-A24 contains the HLA-A24 gene from LB33-MEL.B which wasidentified as allele HLA-A*2402. Following four hours of incubation at37° C., the medium was removed, and replaced by 50 μl of PBS containing10% DMSO. This medium was removed after two minutes and replaced by 200μl of DMEM supplemented with 10% of FCS.

Following this change in medium, COS cells were incubated for 48 hoursat 37° C. Medium was then discarded, and 2000 cells of described CTLclone 269/1 were added, in 100 μl of Iscove's medium containing 10%pooled human serum. Supernatant was removed after 24 hours, and TNFcontent was determined in an assay on WEHI cells, as described byTraversari et al., Immunogenetics 35: 145-152 (1992), the disclosure ofwhich is incorporated by reference.

Of 400 pools tested, one was positive.

EXAMPLE 4

The bacteria of the positive pool were subcloned. Plasmid DNA wasextracted from 600 individual colonies, and cotransfected withpcDNA-I/Amp-A24 into new samples of COS cells in the same manner asdescribed supra, and the cells were again tested for stimulation of CTL269/1. A positive clone was found, identified as "5E10".

The plasmid from the positive clone was removed, and sequenced followingart known techniques.

The sequence identified is 1554 base pairs long (see SEQ ID NO: 1). Thissequence contains an open reading frame encoding 518 amino acids.

The 104 nucleotides at positions 1310-1413 were found to be identical tothe 104 first base pairs of a 113 base pair sequence recorded inGenbank: L25344, HOMRBCESTC "Human (clone 17)" erythroleukemic expressedsequence tag (EST) mRNA fragment. No sequences were found whichcorresponded to the sequence of SEQ ID NO: 1, however.

EXAMPLE 5

Following the isolation of 5E10, described supra, it was used as a probein a standard Northern Blot, using total RNA of LB 33-MEL, and standardtechniques.

The results showed a band of about 2.5 kilobases, which is, of course,somewhat longer than the probe itself. This suggests that clone 5E10 isnot complete.

As a result, the same cDNA library prepared from RNA of LB33-MEL cellswas screened, again using cDNA 5E10 as a probe. A cDNA clone of 2148base pairs was identified, and sequenced. It is referred to as Hi2. Thesequence of 5E10 is completely included in that of Hi2, except that atbase 254, Hi2 has cytosine, while 5E10 has thymine.

EXAMPLE 6

Following the isolation of Hi2, a set of experiments were carried out inorder to confirm that Hi2 was a tumor rejection antigen precursorencoding sequence. Specifically, HLA-A24 positive leukemia cell lineLB804-ALL was used, because prior experiments had shown that CTL 269/17,described supra, did not lyse this line.

Cells of the leukemia line were transfected with expression vectorpEF-BOS-puro.PL3, which carries a gene conferring puromycin resistance,and into which cDNA Hi2 was cloned. Puromycin resistant populations wereselected, and isolated. These proved to be sensitive to CTL 269/17, thusindicating that expression of antigen LB33-E is not dependent upon thehigh copy number which results from COS-7 transfection.

FIGS. 3A and 3B show these results, where 3A shows the leukemia cellline before transfection, where CTL 269/17 is the responder.

The sequence of Hi2 is provided as SEQ ID NO:2. When comparing it toother sequences in data banks, it was found that nucleotides 1486-1589are identical to 104 base pairs of a 113 base pair sequence expressed inmyeloid leukemia cell line K562 (Gen Bank:L25344). Nucleotides 1983-2128are identical to 146 of 147 base pairs expressed in promyelocyticleukemia cell line. HL-60 (DDBJ:D20455), while nucleotides 1736-2067 are97% homologous with 325 base pairs of a 332 base pair cDNA found incells of human testis (Gen Bank:T19428).

Analysis of the sequence of Hi2 shows an open reading frame encoding aputative protein of 509 amino acids, which has no signal sequence. Nosignificant homology was found with other protein sequences in databanks.

EXAMPLE 7

Hi2 was then used to isolate genomic DNA encoding the pertinent protein.The DNA of 12 groups of 70,000 cosmids of a human genomic DNA librarywas collected, and 5E10 was used to hybridize to these, using standardmethodologies. The clone hybridized to one cosmid group. Followingsubcloning one cosmid was identified which hydridized with cDNA clone5E10. The sequence was secured by using primers deduced from the cDNAsequence. The sequence presents six exons, with the open reading framespanning exons 3-6.

EXAMPLE 8

The information in SEQ ID NO: 1 was sufficient to permit analysis ofgene expression via polymerase chain reaction (PCR).

The following primers were used:

    ______________________________________                                        5'-GCCTGCTGAAGGATGAGGCC-3' (SEQ ID NO: 3)                                     5'-GGTGCTGCAGGAGACTCTGC-3' (SEQ ID NO: 4)                                     ______________________________________                                    

These correspond to nucleotides 157-176, and 1328-1347 of SEQ ID NO: 1,respectively.

The PCR was carried out for 28 cycles, (1 cycle: 1 minute 94° C., 2minutes at 65° C., 3 minutes at 72° C.). In carrying out the PCR, 2.5 ulof cDNA template, prepared as described supra, was combined with 2.5 ulof 10×Dynazyme buffer, 0.25 ul of each dNTP (10 mM), 0.5 ul of eachprimer (20 mM), 0.5U Dynazyme (0.25 ul stock, 2 U/ml), and 18.5 ulwater. Table 1, which follows, sets forth the results. Note theexpression over a number of varied tumor samples, as well as tumor celllines, indicating that this is not an artifact of cell culture. Further,with the exception of testis, there is absolutely no expression innormal tissues.

                  TABLE 1                                                         ______________________________________                                        Expression of the gene corresponding to cDNA clone 5E10 in tumors             and normal tissues                                                            Normal tissues:                                                               Liver                    0/1                                                  Stomach                  0/1                                                  Colon                    0/1                                                  Lung                     0/1                                                  Spleen                   0/1                                                  Heart                    0/1                                                  Breast                   0/1                                                  Bladder                  0/1                                                  Prostate                 0/1                                                  Thymus                   0/1                                                  Bone marrow              0/1                                                  Blood lymphocytes        0/1                                                  Fibroblasts              0/1                                                  Testis                   2/2                                                  Tumor samples:                                                                Melanoma                 5/5                                                  Lymphoma                 2/5                                                  Chronic Myeloid Leukemia 1/2                                                  Chronic Lymphoid Leukemia                                                                              1/5                                                  Acute Myeloid Leukemia   0/6                                                  Renal Carcinoma          3/6                                                  Sarcoma                  2/3                                                  Breast carcinoma         2/5                                                  Tumor cell lines:                                                             Melanoma                 11/15                                                Leukemia                 3/6                                                  Burkitt lymphoma         2/4                                                  ______________________________________                                    

EXAMPLE 9

A second assay was carried out, based upon TNF (tumor necrosis factor)release. In this assay, COS-7 cells (10,000 cells/mirowell) weretransfected with the plasmid pcDNAI/Amp carrying HLA-A24 cDNA, asdescribed supra, or cotransfected with this plasmid and plasmidpcDNAI/Amp containing SEQ ID NO: 1, described supra. Twenty four hoursafter transfection, 3000 cells of CTL 269/17 were added to thetransfectants. In a control, the same number of LB33-MEL.B-1 cells wereused. The concentration of TNF released in the cell medium was measuredafter 24 hours, using TNF sensitive cell line WEHI-164c13.

The results are presented in FIG. 4. They show that TNF release by CTLswas provoked only with COS cells cotransfected with vectors expressingHLA-A24 and SEQ ID NO 1. COS cells do not present HlA-A24 on their own,nor do they express the sequences of the invention. When cotransfected,however, they were able to provoke TNF release to a level nearly that ofautologous cell line LB33-MEl.B-1.

The results, as set forth in FIG. 3, not only show that the material ofSEQ ID NO: 1 does in fact code for a tumor rejection antigen precursorwhich stimulates CTLs when processed, it also shows that, as elaboratedupon infra, one can assay for the presence of CTLs which are specificfor tumor cells by suing non-transformed cells, such that the resultingtransfectant will express both HLA-A24 and DAGE.

EXAMPLE 10

As it has been well established that TRAPs are processed to smallertumor rejection antigens, experiments were undertaken to identify atumor rejection antigen or antigens produced from the describedsequences.

The cDNA for 5E10 was partially digested with the endonuclease NsiI, andthe thus truncated cDNA clones were cotransfected into COS-7 cells withHLA-A24 cDNA clones. Transfectants were then tested for expression ofLB33-E, by adding CTL 269/17, and measuring TNF production.

Results are summarized in FIG. 5. Nucleotides corresponding tonucleotides 1047-1260 of the cDNA of Hi2 were found to encode therelevant antigen. Four sequences in this region which (i) were 9 or 10amino acids long, (ii) had Tyr or Phe at position 2, and (iii) had oneof Phe, Leu, Ile, or Trp at C-terminus were possible. This is the motiffor HLA-A24 binding described by Kubo, et al, J. Immunol 152:3913(1994); Meier, et al, Immunogenetics 40:306-308 (1994). These weresynthesized, and incubated with cells of MHC class I negative B-celllympoblastoid line C1R, which had been transfected with the HLA-A24 cDNAclone described supra. One peptide:

Leu Tyr Val Asp Ser Leu Phe Phe Leu (SEQ ID NO. 5), was found tosensitize the transfected C1R-A24 cells to lysis by the anti LB33-ECTLs, with half maximal effect at 500 nM.

Comparative experiments were carried out, wherein peptides containingone additional N- or C-terminal amino acid, and where the C-terminal Leuwas deleted, were used. SEQ ID NO:6 sensitized the cells to lysis,although to a lesser degree than SEQ ID NO:5. SEQ ID NOS:7 and 8 weremuch less sensitive in sensitizing the cells, as is shown in FIG. 6.Peptides used extended SEQ ID NO:5 at the N-terminus with Ala (SEQ IDNO:6), and by deleting the C-terminal Leu (SEQ ID NO:7). Addition of Argto the C terminus resulted in SEQ ID NO:8. In these experiments, ⁵¹ Crlabeled C1R-A24 cells were incubated for 30 minutes in the presence ofindicated peptide concentrations CTLs were added at E/T ratios of 10:1,and chromium release was measured after 4 hours.

EXAMPLE 11

Tests were then carried out, using the well known reverse transcriptasepolymerase chain reaction ("RT-PCR"), to determine expression of thesubject gene. In table 2, which follows the results are shown.

The higher proportion of positive tumors were melanomas (91%), lungsquamous carcinomas (78%), and adenocarcinomas (46%), as well as renalcarcinomas (43%), sarcomas (40%), and acute leukemias (33%).

There was also some expression in normal tissues. Testis, ovary, andendometrium expressed about 10% of what was found in cell line LB33-MEL,while lower levels were found in skin, brain, heart, kidney, and adrenaltissue. Note Table 2 and FIG. 6.

                  TABLE 2                                                         ______________________________________                                        Expression of gene DAGE by tumoral tissues.                                   ______________________________________                                        Tumor samples                                                                 Brain tumors                      1/7                                         Colorectal carcinomas             2/51  4%                                    Gastric carcinomas                1/2                                         Naevi                             9/18                                        Melanomas    primary legions      43/49 88%                                                metastases           144/152                                                                             95%                                                ocular               5/9                                         Neuroblastomas                    2/3                                         Head and neck squamous            17/44 39%                                   carcinomas                                                                    Lung carcinomas                                                                            SCLC                 1/4                                                      NSCLC      adeno-    12/26 46%                                                           carcinomas                                                                    squamous  51/65 78%                                                           carcinomas                                            Prostatic carcinomas              2/20                                        Renal carcinomas                  24/56 43%                                   Bladder tumors                                                                             superficial          4/36  11%                                                infiltrating         9/42  21%                                   Sarcomas                          10/25 40%                                   Mammary carcinomas                45/169                                                                              27%                                   Thyroid carcinomas                3/5                                         Acute leukemias                   21/63 33%                                   Tumor cell lines                                                              Melanomas                         72/74 97%                                   Sarcomas                          4/5                                         Lung carcinomas                                                                            SCLC                 19/27 70%                                                NSCLC                2/2                                         Mesotheliomas                     2/18                                        Head and neck tumors              2/7                                         Bladder tumors                    2/3                                         Colorectal carcinomas             1/15                                        Renal carcinomas                  9/12                                        EBV-transformed lym-              0/8                                         phoblastoid B cell lines                                                      ______________________________________                                    

The foregoing examples show the isolation of a nucleic acid moleculewhich codes for a tumor rejection antigen precursor. This "TRAP" codingmolecule, however, is not homologous with any of the previouslydisclosed MAGE, BAGE or GAGE coding sequences described in thereferences set forth supra. Hence, one aspect of the invention is anisolated nucleic acid molecule having the nucleotide sequence set forthin SEQ ID NO: 1 or SEQ ID NO: 2, as well as those portions of SEQ ID NO:1 or SEQ ID NO:2 which express TRAs such as those encoding SEQ ID NOS:5, 6 and 8 presented by MHC molecules such as HLA-A24, and derived fromDAGE. This sequence is not a MAGE, BAGE or GAGE coding sequence, as willbe seen by comparing it to the sequence of any of these genes asdescribed in the cited references. Also a part of the invention arethose nucleic acid sequences which also code for a non-MAGE, non-BAGEand non-MAGE tumor rejection antigen precursor but which hybridize tothe nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1and/or 2 under stringent conditions. The term "stringent conditions" asused herein refers to parameters with which the art is familiar. Morespecifically, stringent conditions, as used herein, refers tohybridization in 1M NaCl, 1% SDS, and 10% dextran sulfate. This isfollowed by two washes of the filter at room temperature for 5 minutes,in 2×SSC, and one wash for 30 minutes in 2×SSC, 0.1% SDS. There areother conditions, reagents, and so forth which can be used, which resultin the same or higher degree of stringency. The skilled artisanwill befamiliar with such conditions, and, thus, they are not given here.

The widespread distribution in the expression of this gene (7 out of 8types of tumor were found to express it), shows that the isolatednucleic acid molecule can be used as a diagnostic probe to determinepresence of transformed cells. The identification of melanoma was 100%,so on a very basic level, the isolated nucleic acid molecules may beused to determine whether or not melanoma is present. Note that thereare many ways available to the skilled artisan to confirm that a tumorsample is a melanoma sample, and these need not be reiterated here.Further, the rate of success in identifying tumors is in accordance withnucleic acid based diagnostic methods for determining transformation ofcells.

It will also be seen from the examples that the invention embraces theuse of the sequences in expression vectors, which may be used totransform or to transfect host cells and cell lines, be theseprokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO or COS cells). Theexpression vectors require that the pertinent sequence, i.e., thosedescribed supra, be operably linked to a promoter. As it has been foundthat human leukocyte antigen HLA-A24 presents a tumor rejection antigenderived from these genes, the expression vector may also include anucleic acid sequence coding for HLA-A24. In a situation where thevector contains both coding sequences, it can be used to transform ortransfect a cell which does not normally express either one. The tumorrejection antigen precursor coding sequence may be used alone, when,e.g., the host cell already expresses HLA-A24. Of course, there is nolimit on the particular host cell which can be used. As the vectorswhich contain the two coding sequences may be used in HLA-A24 presentingcells if desired, and the gene for tumor rejection antigen precursor canbe used in host cells which do not express HLA-A24.

The invention also embraces so called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

To distinguish the nucleic acid molecules and the TRAPs of the inventionfrom the previously described MAGE, BAGE and GAGE materials, theinvention shall be referred to as the DAGE family of genes and TRAPs.Hence, whenever "DAGE" is used herein, it refers to the tumor rejectionantigen precursors coded for by the previously described sequences."DAGE coding molecule" and similar terms, are used to describe thenucleic acid molecules themselves.

The invention as described herein has a number of uses, some of whichare described herein. First, the invention permits the artisan todiagnose a disorder characterized by expression of the TRAP. Thesemethods involve determining expression of the TRAP gene, and/or TRAsderived therefrom, such as a TRA presented by HLA-A24. In the formersituation, such determinations can be carried out via any standardnucleic acid determination assay, including the polymerase chainreaction, or assaying with labelled hybridization probes. In the lattersituation, assaying with binding partners for complexes of TRA and HLA,such as antibodies, is especially preferred. An alternate method fordetermination is a TNF or ⁵¹ Cr release assay, of the types describedsupra.

The isolation of the TRAP gene also makes it possible to isolate theTRAP molecule itself, especially TRAP molecules containing the aminoacid sequence coded for by SEQ ID NO: 2. These isolated molecules whenpresented as the TRA, or as complexes of TRA and HLA, such as HLA-A24,may be combined with materials such as adjuvants to produce vaccinesuseful in treating disorders characterized by expression of the TRAPmolecule. In addition, vaccines can be prepared from cells which presentthe TRA/HLA complexes on their surface, such as non-proliferative cancercells, non-proliferative transfectants, etcetera. In all cases wherecells are used as a vaccine, these can be cells transfected with codingsequences for one or both of the components necessary to provide a CTLresponse, or be cells which express both molecules without transfection.Further, the TRAP molecule, its associated TRAs, as well as complexes ofTRA and HLA, may be used to produce antibodies, using standardtechniques well known to the art.

When "disorder" is used herein, it refers to any pathological conditionwhere the tumor rejection antigen precursor is expressed. An example ofsuch a disorder is cancer, melanoma in particular. Melanoma is wellknown as a cancer of pigment producing cells.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-A24 cells. One such approach is theadministration of CTLs specific to the complex to a subject withabnormal cells of the phenotype at issue. It is within the skill of theartisan to develop such CTLs in vitro. Specifically, a sample of cells,such as blood cells, are contacted to a cell presenting the complex andcapable of provoking a specific.CTL to proliferate. The target cell canbe a transfectant, such as a COS cell of the type described supra. Thesetransfectants present the desired complex on their surface and, whencombined with a CTL of interest, stimulate its proliferation. COS cells,such as those used herein are widely available, as are other suitablehost cells.

To detail the therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917 (1986); Riddel et al., Science 257:238 (Jul. 10, 1992); Lynch et al., Eur. J. Immunol. 21: 1403-1410(1991); Kast et al., Cell 59: 603-614 (Nov. 17, 1989)), cells presentingthe desired complex are combined with CTLs leading to proliferation ofthe CTLs specific thereto. The proliferated CTLs are then administeredto a subject with a cellular abnormality which is characterized bycertain of the abnormal cells presenting the particular complex, wherethe complex contains the pertinent HLA molecule. The CTLs then lyse theabnormal cells, thereby achieving the desired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/TRA complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a DAGE sequence. Once cells presenting the relevant complex areidentified via the foregoing screening methodology, they can be combinedwith a sample from a patient, where the sample contains CTLs. If thecomplex presenting cells are lysed by the mixed CTL sample, then it canbe assumed that a GAGE derived, tumor rejection antigen is beingpresented, and the subject is an appropriate candidate for thetherapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach, i.e., the use of non-proliferativecells expressing the complex, has been elaborated upon supra. The cellsused in this approach may be those that normally express the complex,such as irradiated melanoma cells or cells transfected with one or bothof the genes necessary for presentation of the complex. Chen et al.,Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies thisapproach, showing the use of transfected cells expressing HPV E7peptides in a therapeutic regime. Various cell types may be used.Similarly, vectors carrying one or both of the genes of interest may beused. Viral or bacterial vectors are especially preferred. In thesesystems, the gene of interest is carried by, e.g., a Vaccinia virus orthe bacteria BCG, and the materials de facto "infect" host cells. Thecells which result present the complex of interest, and are recognizedby autologous CTLs, which then proliferate. A similar effect can beachieved by combining the tumor rejection antigen or the precursoritself with an adjuvant to facilitate incorporation into HLA-A24presenting cells which then present the HLA/peptide complex of interest.The TRAP is processed to yield the peptide partner of the HLA moleculewhile the TRA is presented without the need for further processing.

Also a feature of this invention are isolated peptides derived from theDAGE TRAP which conform to the rules for presentation by MHC molecules.For example, in PCT application No. PCT/US93/07421, incorporated byreference herein, several motifs are described as being associated withdifferent MHC molecules. These motifs, incorporated by reference herein,as well as those taught by, e.g. Falk et al., Nature 351: 290-296(1991); Engelhard, Ann. Rev. Immunol 12: 181-207 (1994); Ruppert et al.,Cell 74: 929-937 (1993); Rotzschke et al., Nature 348: 252-254 (1990);Bjorkman et al., Nature 329: 512-518 (1987) and Traversari et al., J.Exp. Med. 176: 1453-1457 (1992) all of which are incorporated byreference, serve as a basis for identifying appropriate peptidesobtainable or derivable from the DAGE gene. These peptides may be usedalone, or in mixtures, in another aspect of the invention, which is nowdescribed. Exemplary of these are the following. For HLA-A2, a bindingmotif is Xaa Leu Xaa Gly (Xaa)_(n) Leu (SEQ ID NO: 9) where n is 4 or 5.Amino acids 100-108 of SEQ ID NO: 1 correspond to this motif. A secondmotif for HLA-A2 replaces terminal Leu with Val (SEQ ID NO: 16), andamino acids 355-364 satisfy this motif. For HLA-A3, the motifs are XaaLeu (Xaa)₆ (Lys or Tyr) (SEQ ID NO:10 and SEQ ID NO: 11). Amino acids48-56, 100-108, 138-146, 214-222, and 257-265 of SEQ ID NO: 17 wellsatisfy this motif. For HLA-A11, the motif (Xaa)₇ Lys Lys (SEQ ID NO:12) is known, and amino acids 150-158, 195-203, and 204-212 will satisfyit. For HLA-A24, the known motif is Xaa Tyr (Xaa)₆ Leu, (SEQ ID NO: 18)and is satisfied by amino acids 254-262, and 447-455 as well as SEQ IDNO: 5. For HLA-B7, the motif is Xaa Pro Arg (Xaa)₅ Leu, (SEQ ID NO: 14)and amino acids 48-56 meet it. For HLA-B8, (Xaa)₂ Lys Xaa Lys (Xaa)₃ Leu(SEQ ID NO: 14) is the motif, satisfied by amino acids 156-164 and198-206. For HLA-B44, motif Xaa Glu (Xaa)₃ Asp (Xaa)₂ Phe (SEQ ID NO:15) is satisfied by amino acids 184-192. For HLA-Cw*1601 binding issatisfied by amino acids 40-48, and 375-383.

The fact that a number of sequences are present which correspond to HLAmotifs suggests what will be referred to herein as "cocktail"therapeutic and diagnostic uses. It is expected that in a typical CTLresponse to tumor cells, CTLs specific to more than one complex ofpeptide and HLA molecule will proliferate. For example, it may be thecase that for HLA-A24 presenting cells, CTLs specific for HLA-A24 andamino acid sequence 467-475 will proliferate. Thus, one ca optimize theassay by using both peptides when attempting to identify CTLs.Similarly, the therapeutic methods might be optimized by using more thanone HLA-A24 binding peptide.

It is well known that individuals are not "monovalent" for HLAmolecules, as cells present more than one kind of HLA. Thus, one canmaximize diagnostic and/or therapeutic by combining a number of peptidesas described supra in a diagnostic assay to determine CTLs, or to treatpatients in the therapies described supra.

Any concern as to false positives, is believed to be misplaced because,as noted supra, the nucleic acid molecules of the invention have beenfound to be expressed only in tumor cells, so the presence of CTLs tothe HLA and the peptides must be considered de facto evidence of thepresence, at some time in the past of the present existence of acancerous or transformed condition. Thus, cocktails of the peptides ofthe invention can be prepared. Determination of the components of themixture is not difficult, because all that is needed is one or more ofthe HLA types presented by the individual under consideration. HLAtyping is a very standard technique, well known in the art; and wellwithin the abilities and skill of the artisan.

It has been fairly well established that the blood of individualsafflicted with tumors frequently contains cytolytic T cells ("CTLs")against complexes of MHC molecules and presented peptides. See, e.g.,Robbins et al., Canc. Res. 54: 3124-3126 (1994); Topolian et al., J.Immunol. 142: 3714-3725 (1989); Coulie et al., Int. J. Cancer 50:289-297 (1992), all of which are incorporated by reference. Also, noteKawakami et al., J. Exp. Med. 180: 347-352 (1994); Hom et al., J.Immunother 10: 153-164 (1991), Darrow et al, J. Immunol. 142(9):3329-3335 (1989); Slovin et al., J. Immunol. 137(9): 3042-3048 (1986),all of which are incorporated by reference. These papers all establishthe usefulness of a CTL proliferation assay to diagnose cancer.Expressed generally, one takes a peripheral blood lymphocyte (PBL)containing sample from a subject to be tested. Assuming that the patientdoes have a tumor, or the subject's cells have began to undergotransformation, CTLs which are specific to transformed cells with becontained in that sample. These CTLs can be stimulated to proliferatevia contact with a target cell which presents complexes of a relevantMHC molecule and the peptide presented thereby. For example, as wasshown, supra, DAGE derived tumor rejection antigens ("TRAs") arepresented by HLA-A24 cells. Thus, by mixing the PBL sample with a targetof HLA-A24 presenting cells and peptides which are derived from a TRAPand presented by HLA-A24, one can observe CTL proliferation, and thusdiagnose for the presence of transformed cells. These cells can be cellswhich normally present the MHC molecule in question, but can also becells transformed by an HLA coding sequence. The cells may be tumorcells, or normal cells. Various ways of determining CTL proliferationare known, including TNF release assays, and ⁵¹ Cr release assays. Othermethodologies are also available. Thus, one aspect of the inventioninvolves mixing a target cell sample with a peptide or mix of peptidesderived from a DAGE TRA and presented by the MHC molecules of the targetcell sample and with the PBLs of the subject under evaluation. Themixture is then tested for CTL proliferation.

The peptide or peptides may also be combined with one or more adjuvantswhich stimulate a more pronounced CTL response. Exemplary of suchadjuvants are saponins and their derivatives, such as those disclosed byU.S. Pat. No. 5,057,540 to Kensil et al., incorporated by reference orPCT application PCT/US92/03579 to Scott et al., also incorporated byreference. Of course, standard adjuvants, such as Freund's completeadjuvant, or Freund's incomplete adjuvant, may also be used.

Other aspects of the invention will be clear to the skilled artisan andneed not be repeated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES:  18                                           - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    15 - #54 base pairs                                  # nucleic acidTYPE:                                                                     (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: nucleic acid                                        -     (ix) FEATURE:                                                           #(5E10)   (A) NAME/KEY:  DAGE                                                 #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              50AATCCA AGCGTTGGAG GTCCTGAGGC CAGCCTAAGT                      #             100ACGAAG GCGTTTGCGG GGTTCCATTC AGAGCCGATA                      #             150TGTGGA CAAGCCCACG GAGACTTGTG GAGCTGGCAG                      #             200AAGGAT GAGGCCCTGG CCATTGCCGC CCTGGAGTTG                      #             250CTTCCC GCCACTCTTC ATGGCAGCCT TTGACGGGAG                      #             300TGAAGG CAATGGTGCA GGCCTGGCCC TTCACCTGCC                      #             350CTGATG AAGGGACAAC ATCTTCACCT GGAGACCTTC                      #             400TGGACT TGATGTGCTC CTTGCCCAGG AGGTTCGCCC                      #             450TTCAAG TGCTGGATTT ACGGAAGAAC TCTCATCAGG                      #             500TGGTCT GGAAACAGGG CCAGTCTGTA CTCATTTCCA                      #             550TCAGCC CATGACAAAG AAGCGAAAAG TAGATGGTTT                      #             600AGCAGC CCTTCATTCC AGTAGAGGTG CTCGTAGACC                      #             650GGTGCC TGTGATGAAT TGTTCTCCTA CCTCATTGAG                      #             700GAAAAA TGTACTACGG CTGTGCTGTA AGAAGCTGAA                      #             750CCATGC AGGATATCAA GATGATCCTG AAAATGGTGC                      #             800GAAGAT TTGGAAGTGA CTTGTACCTG GAAGCTACCC                      #             850TTCTCC TTACCTGGGC CAGATGATTA ATCTGCGTAG                      #             900ACATCC ATGCATCTTC CTACATTTCC CCGGAGAAGG                      #             950GCCCAG TTCACCTCTC AGTTCCTCAG TCTGCAGTGC                      #            1000TGTGGA CTCTTTATTT TTCCTTAGAG GCCGCCTGGA                      #            1050ACGTGA TGAACCCCTT GGAAACCCTC TCAATAACTA                      #            1100GAAGGG GATGTGATGC ATCTGTCCCA GAGTCCCAGC                      #            1150TGTCCT GAGTCTAAGT GGGGTCATGC TGACCGATGT                      #            1200TCCAAG CTCTGCTGGA GAGAGCCTCT GCCACCCTCC                      #            1250GATGAG TGTGGGATCA CGGATGATCA GCTCCTTGCC                      #            1300GAGCCA CTGCTCCCAG CTTACAACCT TAAGCTTCTA                      #            1350CCATAT CTGCCTTGCA GAGTCTCCTG CAGCACCTCA                      #            1400CTGACC CACGTGCTGT ATCCTGTCCC CCTGGAGAGT                      #            1450TGGTAC CCTCCACCTG GAGAGGCTTG CCTATCTGCA                      #            1500AGTTGC TGTGTGAGTT GGGGCGGCCC AGCATGGTCT                      #            1550CCCTGT CCTCACTGTG GGGACAGAAC CTTCTATGAC                      #           1554                                                              - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    21 - #48 base pairs                                  # nucleic acidTYPE:                                                                     (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: nucleic acid                                        -     (ix) FEATURE:                                                           #(Hi2)    (A) NAME/KEY:  DAGE                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #              50CTCCCC CGCAGCCAGA AGCCGGGCCT GCAGCCCCTC                      #             100ACACCC CACCCGCTTC CCAGGCGTGA CCTGTCAACA                      #             150TGGTGA ACTCTCTGAG GAAAAACCAT TTTGATTATT                      #             200GTGGCA ACAAGTGACT GAGACCTAGA AATCCAAGCG                      #             250GCCAGC CTAAGTCGCT TCAAAATGGA ACGAAGGCGT                      #             300TCAGAG CCGATACATC AGCATGAGTG TGTGGACAAG                      #             350TGGAGC TGGCAGGGCA GAGCCTGCTG AAGGATGAGG                      #             400GCCCTG GAGTTGCTGC CCAGGGAGCT CTTCCCGCCA                      #             450CTTTGA CGGGAGACAC AGCCAGACCC TGAAGGCAAT                      #             500CCTTCA CCTGCCTCCC TCTGGGAGTG CTGATGAAGG                      #             550CTGGAG ACCTTCAAAG CTGTGCTTGA TGGACTTGAT                      #             600GGAGGT TCGCCCCAGG AGGTGGAAAC TTCAAGTGCT                      #             650ACTCTC ATCAGGACTT CTGGACTGTA TGGTCTGGAA                      #             700TACTCA TTTCCAGAGC CAGAAGCAGC TCAGCCCATG                      #             750AGTAGA TGGTTTGAGC ACAGAGGCAG AGCAGCCCTT                      #             800TGCTCG TAGACCTGTT CCTCAAGGAA GGTGCCTGTG                      #             850TACCTC ATTGAGAAAG TGAAGCGAAA GAAAAATGTA                      #             900TAAGAA GCTGAAGATT TTTGCAATGC CCATGCAGGA                      #             950TGAAAA TGGTGCAGCT GGACTCTATT GAAGATTTGG                      #            1000TGGAAG CTACCCACCT TGGCGAAATT TTCTCCTTAC                      #            1050TAATCT GCGTAGACTC CTCCTCTCCC ACATCCATGC                      #            1100CCCCGG AGAAGGAAGA GCAGTATATC GCCCAGTTCA                      #            1150AGTCTG CAGTGCCTGC AGGCTCTCTA TGTGGACTCT                      #            1200AGGCCG CCTGGATCAG TTGCTCAGGC ACGTGATGAA                      #            1250TCTCAA TAACTAACTG CCGGCTTTCG GAAGGGGATG                      #            1300CAGAGT CCCAGCGTCA GTCAGCTAAG TGTCCTGAGT                      #            1350GCTGAC CGATGTAAGT CCCGAGCCCC TCCAAGCTCT                      #            1400CTGCCA CCCTCCAGGA CCTGGTCTTT GATGAGTGTG                      #            1450CAGCTC CTTGCCCTCC TGCCTTCCCT GAGCCACTGC                      #            1500CTTAAG CTTCTACGGG AATTCCATCT CCATATCTGC                      #            1550TGCAGC ACCTCATCGG GCTGAGCAAT CTGACCCACG                      #            1600CCCCTG GAGAGTTATG AGGACATCCA TGGTACCCTC                      #            1650TGCCTA TCTGCATGCC AGGCTCAGGG AGTTGCTGTG                      #            1700CCAGCA TGGTCTGGCT TAGTGCCAAC CCCTGTCCTC                      #            1750ACCTTC TATGACCCGG AGCCCATCCT GTGCCCCTGT                      #            1800GCTGGG TGCACATATC AAATGCTTCA TTCTGCATAC                      #            1850CAGGAT GTGCATGCAT CTTGAAGCAA CAAAGCAGCC                      #            1900ATGTTC AGTGTGAGTG AGGAAAACAT GTTCAGTGAG                      #            1950ACAAAT GTTCAGTGAG GAAAAAAAGG GGAAGTTGGG                      #            2000GACTTG AGGAGTTAAT GTGATCTTTG GGGAGATACA                      #            2050AAATAG AATCTGAATT TCTAAAGGGA GATTCTGGCT                      #            2100AGGAGT TAATCCCTGT GTAGACTGTT GTAAAGAAAC                      #              2148AAGC AATGTGAAGC AAAAAAAAAA AAAAAAAA                        - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    20 - # base pairs                                    # nucleic acidTYPE:                                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: nucleic acid                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY:  PCR p - #rimer                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 # 20               GGCC                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    20 - # base pairs                                    # nucleic acidTYPE:                                                                     (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: nucleic acid                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY:  PCR p - #rimer                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 # 20               CTGC                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #peptide  (A) NAME/KEY:  DAGE                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Leu Tyr Val Asp Ser Leu Phe Phe Leu                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    10 - # amino acids                                   # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #peptide  (A) NAME/KEY:  DAGE                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Ala Leu Tyr Val Asp Ser Leu Phe Phe Leu                                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    8                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #peptide  (A) NAME/KEY:  DAGE                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Leu Tyr Val Asp Ser Leu Phe Phe                                                             5                                                             - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    10 - # amino acids                                   # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #peptide  (A) NAME/KEY:  DAGE                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Leu Tyr Val Asp Ser Leu Phe Phe Leu Arg                                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    10 - # amino acids                                   # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-A2                                               # One of the Xaa's between Leu and                                            #may be absent   Gly                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Xaa Leu Xaa Xaa Xaa Xaa Xaa Gly Xaa Leu                                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-A3                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Lys                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-A3                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Tyr                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-A11                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Lys                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-B7                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Xaa Pro Arg Xaa Xaa Xaa Xaa Xaa Leu                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-B8                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Xaa Xaa Lys Xaa Lys Xaa Xaa Xaa Leu                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-B44                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Xaa Glu Xaa Xaa Xaa Asp Xaa Xaa Phe                                                         5                                                             - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    10 - # amino acids                                   # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-A2                                               #  One Xaa between Leu and Gly may be                                                        absent                                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Xaa Leu Xaa Xaa Xaa Xaa Xaa Gly Xaa Val                                                      5                                                            - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:    50 - #9 amino acids                                  # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #amino acid sequenceEY:  DAGE                                                                correspondin - #g to SEQ ID NO:2                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Met Glu Arg Arg Arg Leu Trp Gly Ser Ile Gl - #n Ser Arg Tyr Ile Ser         #15                                                                           - Met Ser Val Trp Thr Ser Pro Arg Arg Leu Va - #l Glu Leu Ala Gly Gln         #             30                                                              - Ser Leu Leu Lys Asp Glu Ala Leu Ala Ile Al - #a Ala Leu Glu Leu Leu         #         45                                                                  - Pro Arg Glu Leu Phe Pro Pro Leu Phe Met Al - #a Ala Phe Asp Gly Arg         #    605                                                                      - His Ser Gln Thr Leu Lys Ala Met Val Gln Al - #a Trp Pro Phe Thr Cys         # 80                                                                          - Leu Pro Leu Gly Val Leu Met Lys Gly Gln Hi - #s Leu His Leu Glu Thr         #                 95                                                          - Phe Lys Ala Val Leu Asp Gly Leu Asp Val Le - #u Leu Ala Gln Glu Val         #           110                                                               - Arg Pro Arg Arg Trp Lys Leu Gln Val Leu As - #p Leu Arg Lys Asn Ser         #       125                                                                   - His Gln Asp Phe Trp Thr Val Trp Ser Gly As - #n Arg Ala Ser Leu Tyr         #   140                                                                       - Ser Phe Pro Glu Pro Glu Ala Ala Gln Pro Me - #t Thr Lys Lys Arg Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Asp Gly Leu Ser Thr Glu Ala Glu Gln Pr - #o Phe Ile Pro Val Glu         #               175                                                           - Val Leu Val Asp Leu Phe Leu Lys Glu Gly Al - #a Cys Asp Glu Leu Phe         #           190                                                               - Ser Tyr Leu Ile Glu Lys Val Lys Arg Lys Ly - #s Asn Val Leu Arg Leu         #       205                                                                   - Cys Cys Lys Lys Leu Lys Ile Phe Ala Met Pr - #o Met Gln Asp Ile Lys         #   220                                                                       - Met Ile Leu Lys Met Val Gln Leu Asp Ser Il - #e Glu Asp Leu Glu Val         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Cys Thr Trp Lys Leu Pro Thr Leu Ala Ly - #s Phe Ser Pro Tyr Leu         #               255                                                           - Gly Gln Met Ile Asn Leu Arg Arg Leu Leu Le - #u Ser His Ile His Ala         #           270                                                               - Ser Ser Tyr Ile Ser Pro Glu Lys Glu Glu Gl - #n Tyr Ile Ala Gln Phe         #       285                                                                   - Thr Ser Gln Phe Leu Ser Leu Gln Cys Leu Gl - #n Ala Leu Tyr Val Asp         #   300                                                                       - Ser Leu Phe Phe Leu Arg Gly Arg Leu Asp Gl - #n Leu Leu Arg His Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Met Asn Pro Leu Glu Thr Leu Ser Ile Thr As - #n Cys Arg Leu Ser Glu         #               335                                                           - Gly Asp Val Met His Leu Ser Gln Ser Pro Se - #r Val Ser Gln Leu Ser         #           350                                                               - Val Leu Ser Leu Ser Gly Val Met Leu Thr As - #p Val Ser Pro Glu Pro         #       365                                                                   - Leu Gln Ala Leu Leu Glu Arg Ala Ser Ala Th - #r Leu Gln Asp Leu Val         #   380                                                                       - Phe Asp Glu Cys Gly Ile Thr Asp Asp Gln Le - #u Leu Ala Leu Leu Pro         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Leu Ser His Cys Ser Gln Leu Thr Thr Le - #u Ser Phe Tyr Gly Asn         #               415                                                           - Ser Ile Ser Ile Ser Ala Leu Gln Ser Leu Le - #u Gln His Leu Ile Gly         #           430                                                               - Leu Ser Asn Leu Thr His Val Leu Tyr Pro Va - #l Pro Leu Glu Ser Tyr         #       445                                                                   - Glu Asp Ile His Gly Thr Leu His Leu Glu Ar - #g Leu Ala Tyr Leu His         #   460                                                                       - Ala Arg Leu Arg Glu Leu Leu Cys Glu Leu Gl - #y Arg Pro Ser Met Val         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Trp Leu Ser Ala Asn Pro Cys Pro His Cys Gl - #y Asp Arg Thr Phe Tyr         #               495                                                           - Asp Pro Glu Pro Ile Leu Cys Pro Cys Phe Me - #t Pro Asn                     #           505                                                               - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #amino acids) LENGTH:    9                                                    # amino acid) TYPE:                                                                     (D) TOPOLOGY:  linear                                               -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                           #binding motifNAME/KEY:  HLA-A27                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Leu                                         __________________________________________________________________________

We claim:
 1. A method for determining possible presence of cancer in asample, comprising assaying said sample to determining expression of agene, cDNA of which hybridizes to the complement of the nucleotidesequence set forth in SEQ ID NO: 1, under stringent conditions bycontacting said sample with a nucleotide sequence which specificallyhybridizes to mRNA or cDNA for said gene, wherein hybridization of saidnucleotide sequence to said mRNA or cDNA is indicative of possiblepresence of cancer in said sample.
 2. The method of claim 1, whereinsaid cDNA comprises the nucleotide sequence set forth in SEQ ID NO: 1,or SEQ ID NO:
 2. 3. The method of claim 1, wherein said cancer isleukemia.
 4. The method of claim 1, wherein said cancer is melanoma,lymphoma, renal carcinoma, sarcoma, or breast carcinoma.
 5. The methodof claim 1, wherein said cancer is lung squamous carcinoma or lungadenocarcinoma.
 6. The method of claim 1, wherein said leukemia is acuteleukemia.
 7. The method of claim 1, wherein said cancer is naevi,neuroblastoma, head and neck squamous carcinoma, bladder cancer, orthyroid carcinoma.